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Biomedical subjects

I M Verma

Publications and source records attributed to I M Verma.

At least 217 records · Page 12Linked to original sources
PubMed1982

Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: expression and regulation.

We have introduced the rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector. The ability of the viral DNA to induce foci in the recipient cells was used as a dominant selection marker. Several copies of rat growth hormone DNA were integrated in the mouse cells. The transformed mouse cells expressed rat growth hormone-specific mRNA and secreted mature rat growth hormone. In rat cells, the expression of this gene is regulated by glucocorticoids. We demonstrate that hormone-dependent regulation transfers with the clone and thus appears to be an intrinsic property of the gene or its RNA products.

Animals↗
PubMed1982

Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed.

AKR murine leukemia virus↗
PubMed1982

Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.

Animals↗
PubMed1982

FBJ murine osteosarcoma virus: identification and molecular cloning of biologically active proviral DNA.

A 12.0-kilobase EcoRI restriction fragment containing FBJ murine osteosarcoma virus (FBJ-MSV) proviral DNA was identified in FBJ-MSV-transformed nonproducer rat cells and molecularly cloned in bacteriophage Charon 30 (lambda FBJ-1). A 5.8-kb HindIII fragment containing the entire FBJ-MSV proviral DNA was isolated from lambda FBJ-1 and subsequently subcloned in plasmid pBR322 (pFBJ-2). The DNA from recombinant plasmid pFBJ-2 was able to induce morphological transformation of rat fibroblasts in tissue culture. Transfected cells contained the p55 and p39 antigens specific for cells transformed by FBJ-MSV (T. Curran and N. M. Teich, J. Virol. 42:114-122, 1982). The organization of the FBJ-MSV provirus was analyzed by restriction endonuclease mapping, and a region of nonhomology with the helper virus was delineated. Sequences specific for this region (presumably the viral fos gene) were subcloned and used as a probe to identify related sequences present in the normal genomes of cells from a variety of mammalian species (cellular fos). A single-size (3.4 kilobases long) class of RNA hybridizing to the viral fos probe was identified in FBJ-MSV-transformed cells.

Animals↗
PubMed1981

Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus.

The complete nucleotide sequence of the transforming gene of a mouse sarcoma virus has been determined. It codes for a protein of 374 amino acids. The nucleotide sequence of the junctions between a murine leukaemia virus and cellular sequences leading to the formation of the viral transforming gene have also been elucidated. The viral transforming sequence and its cellular homologue share an uninterrupted stretch of 1,159 nucleotides, with few base substitutions. The predicted amino acid sequence of the mouse sarcoma virus transforming gene was found to share considerable homology with the proposed amino acid sequence of the avian sarcoma virus oncogene (src) product.

Amino Acid Sequence↗
PubMed1981

M-MuLV-induced leukemogenesis: integration and structure of recombinant proviruses in tumors.

M-MuLV-specific DNA probes were used to establish the state of integration and amplification of recombinant proviral sequences in Moloney virus-induced tumors of Balb/Mo, Balb/c and 129 mice. The somatically acquired viral sequences contain both authentic M-MuLV genomes and recombinants of M-MuLV with endogenous viral sequences. All reintegrated genomes carry long terminal repeat (LTR) sequences at both termini of their genome. In the preleukemic stage a large population of cells exhibiting a random distribution of reintegrated M-MuLV genomes are seen, but during outgrowth of the tumor, selection of cells occurs leaving one or a few clonal descendants in the outgrown tumor. In this latter stage recombinant genomes can be detected. Although these recombinants constitute a heterogeneous group of proviruses, characteristic molecular markers are conserved among many individual proviral recombinants, lending credence to the notion that a certain recombinant structure is a prerequisite for the onset of neoplasia. The structure of these recombinants shows close structural similarities to the previously described mink cell focus-inducing (MCF)-type viruses.

Animals↗
PubMed1981

Analysis of transforming gene products from Moloney murine sarcoma virus.

We previously showed that in vitro translation of M-MuSV virion RNA yielded a 62 kd gag gene product and an overlapping set of four proteins with approximate molecular weights of 37,000, 33,000, 24,000 and 18,000. In this paper we show, by use of hybrid arrest translation with cloned recombinant DNAs containing M-MuSV v-mosMo sequences, that the 37, 33, 24 and 18 kd proteins are synthesized in their entirety from the v-mosMo gene. Analysis of the primary sequence of these proteins shows that each one is initiated independently from AUG codons within the v-mosMo gene and utilizes the long open reading frame predicted from the v-mosMo DNA sequence. Antisera against synthetic peptides corresponding to the C terminus of the predicted v-mosMo gene product precipitate all four in vitro v-mosMo proteins.

Amino Acid Sequence↗
PubMed1981

Identification of a RNA polymerase II initiation site in the long terminal repeat of Moloney murine leukemia viral DNA.

We have used a soluble in vitro RNA polymerase II transcription system to define the site of initiation of Moloney murine leukemia viral RNA synthesis. Molecularly cloned integrated and unintegrated Moloney murine leukemia virus DNAs were used as templates. The 5' ends of in vitro transcripts and virion RNA of Moloney murine leukemia virus were compared by nuclease S1 protection experiments. Our results indicate that viral sequences upstream of the in vivo cap site are implicated in the transcription of viral RNA and that the 5' end of an in vitro transcript derived from an integrated Moloney murine leukemia virus clone corresponds to the 5' end of viral genomic RNA.

Cell-Free System↗
PubMed1980

Molecular cloning of unintegrated Moloney mouse sarcoma virus DNA in bacteriophage lambda.

The covalently closed circular forms of unintegrated viral DNA obtained from cells infected with Moloney mouse sarcoma virus was cloned in bacteriophage lambda. The viral DNA was cleaved with restriction endonuclease HindIII and inserted in the unique HindIII site of lambda Charon 21A DNA. Recombinant clones containing virus-reactive DNA sequences were analyzed by restriction endonuclease mapping, R-loop formation, and infectivity assays. Two of eight genome-length recombinant clones characterized contained the large terminal repeat. Only the recombinant clones containing the large terminal repeat were able to induce focus formation in uninfected mouse fibroblasts.

Animals↗
PubMed1980

Identification and molecular cloning of Moloney mouse sarcoma virus-specific sequences from uninfected mouse cells.

When uninfected mouse cell DNA is cleaved with restriction endonuclease EcoRI, a DNA fragment of 14.0 kilobases can be identified by hybridization to cloned DNA containing sarcoma specific sequences of Moloney mouse sarcoma virus (M-MSVsrc). The cellular DNA fragment contains the entire M-MSVsrc specific sequences. The 14.0-kilobase EcoRI DNA fragment was cloned in bacteriophage lambda. The sequence organization of a recombinant clone, lambda . MTX-1, was analyzed by restriction endonuclease mapping, nuclease S1 mapping, and electron microscopy. The results indicate that lambda . MTX-1 contains an uninterrupted stretch of 1.0 kilobase similar to that found in the M-MSV genome.

Animals↗
PubMed1980

Nucleotide sequence of Moloney leukemia virus: 3' end reveals details of replications, analogy to bacterial transposons, and an unexpected gene.

We have determined the sequence of a cloned DNA fragment 1108 base pairs long which corresponds to the 3' end of the Moloney murine leukemia provirus. The clone was obtained as the primary product of reverse transcription and begins with the Moloney "strong stop" sequence, then extends towards the 5' end of the provirus. Our sequence: (i) proves that reverse transcriptase switches templates during minus strand synthesis; (ii) defines the limits of the 515-base-pair repeats which occupy both ends of the integrated provirus; (iii) shows that the structure of the proviral repeats has strong analogy to bacterial insertion sequences, indicating that the Moloney provirus is a transposon; (iv) identifies the putative promotor for genomic transcription within these repeats; (v) shows that the presumed origin of second strand synthesis, which lies just outside the 3' repeat, has tertiary structure analogous to single-stranded bacteriophage origins of replication; (vi) solves the amino acid sequence of most of pI5E, the carboxy-terminal product of the env gene; (vii) allows detailed mapping of the mink cell focus-forming virus substitution locus in a central location within the gp70 region of the env gene; and (viii) identifies a long open translation frame to the right of the env gene (R gene) which could be involved in leukemogenesis.

Animals↗
PubMed1980

Structure of Moloney murine leukemia viral DNA: nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences.

Some unintegrated and all integrated forms of murine leukemia viral DNA contain long terminal repeats (LTRs). The entire nucleotide sequence of the LTR and adjacent cellular sequences at the 5' end of a cloned integrated proviral DNA obtained from BALB/Mo mouse has been determined. It was compared to the nucleotide sequence of the LTR at the 3' end. The results indicate: (i) a direct 517-nucleotide repeat at the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end of the primer tRNA (strong-stop DNA); (iii) an 11-nucleotide inverted repeat is present at the ends of the 5'-LTR and a total of 17 out of 21 nucleotides at the termini are inverted repeats; (iv) sequences CAATAAAAG (at positions -24 to -31) and CAATAAAC (at positions +46 to +53) resembling the hypothetical DNA-dependent RNA polymerase II promoter site can be identified in the 5'-LTR; (v) the sequence GAAA appears to be repeated on both sides of the junction of viral and cellular sequences; and (vi) in analogy with the bacterial transposons, the presence of an inverted repeat sequence at the termini of 5'-LTR suggests that M-MLV also has the integration properties of a transposon.

Animals↗